Sleep-wake control is dependent upon multiple brain areas widely distributed throughout the neural axis. Historically, the monoaminergic and cholinergic neurons of the ascending arousal system were the first to be discovered, and it was only relatively recently that GABAergic and glutamatergic wake- and sleep-promoting populations have been identified. Contemporary advances in molecular-genetic tools have revealed both the complexity and heterogeneity of GABAergic NREM sleep-promoting neurons as well as REM sleep-regulating populations in the brainstem such as glutamatergic neurons in the sublaterodorsal nucleus. The sleep-wake cycle progresses from periods of wakefulness to non-rapid eye movement (NREM) sleep and subsequently rapid eye movement (REM) sleep. Each vigilance stage is controlled by multiple neuronal populations, via a complex regulation that is still incompletely understood. In recent years the field has seen a proliferation in the identification and characterization of new neuronal populations involved in sleep-wake control thanks to newer, more powerful molecular genetic tools that are able to reveal neurophysiological functions via selective activation, inhibition and lesion of neuroanatomically defined sub-types of neurons that are widespread in the brain, such as GABAergic and glutamatergic neurons.1,2.

Aging and Alzheimers disease (AD) are both associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Slow-wave-sleep deficits have been shown to worsen AD symptoms and prevent healthy aging. However, the mechanism remains poorly understood due to the lack of animal models in which SWS can be specifically manipulated. Notably, a mouse model of SWS enhancement has been recently developed in adult mice. As a prelude to studies assessing the impact of SWS enhancement on aging and neurodegeneration, we first asked whether SWS can be enhanced in animal models of aging and AD. The chemogenetic receptor hM3Dq was conditionally expressed in GABAergic neurons of the parafacial zone of aged mice and AD (APP/PS1) mouse model. Sleep-wake phenotypes were analyzed in baseline condition and following clozapine-N-oxide (CNO) and vehicle injections. Both aged and AD mice display deficits in sleep quality, characterized by decreased slow wave activity. Both aged and AD mice show SWS enhancement following CNO injection, characterized by a shorter SWS latency, increased SWS amount and consolidation, and enhanced slow wave activity, compared with vehicle injection. Importantly, the SWS enhancement phenotypes in aged and APP/PS1 model mice are comparable to those seen in adult and littermate wild-type mice, respectively. These mouse models will allow investigation of the role of SWS in aging and AD, using, for the first time, gain-of SWS experiments.

Parafacial zone (PZ) GABAergic neurons play a major role in slow-wave-sleep (SWS), also called non-rapid eye movement (NREM) sleep. The PZ also contains glutamatergic neurons expressing the vesicular transporter for glutamate, isoform 2 (Vglut2). We hypothesized that PZ Vglut2-expressing (PZVglut2) neurons are also involved in sleep control, playing a synergistic role with PZ GABAergic neurons. To test this hypothesis, we specifically activated PZVglut2 neurons using the excitatory chemogenetic receptor hM3Dq. Anatomical inspection of the injection sites revealed hM3Dq transfection in PZ, parabrachial nucleus (PB), sublaterodorsal nucleus (SLD) or various combinations of these three brain areas. Consistent with the known wake- and REM sleep-promoting role of PB and SLD, respectively, chemogenetic activation of PBVglut2 or SLDVglut2 resulted in wake or REM sleep enhancement. Chemogenetic activation of PZVglut2 neurons did not affect sleep-wake phenotype during the mouse active period but increased wakefulness and REM sleep, similar to PBVglut2 and SLDVglut2 activation, during the rest period. To definitively confirm the role of PZVglut2 neurons, we used a specific marker for PZVglut2 neurons, Phox2B. Chemogenetic activation of PZPhox2B neurons did not affect sleep-wake phenotype, indicating that PZ glutamatergic neurons are not sufficient to affect sleep-wake cycle. These results indicate that PZ glutamatergic neurons are not involved in sleep-wake control.

Intrinsically photoreceptive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and convey retinal light inputs to the circadian system via the retinohypothalamic tract (RHT) projection to the suprachiasmatic nucleus (SCN). The principal neurotransmitter of this projection is glutamate, and ipRGCs use the vesicular glutamate transporter 2 (VGLUT2) to package glutamate into synaptic vesicles. However, these neurons contain other potential neurotransmitters, such as pituitary adenylate cyclase activating polypeptide (PACAP). To test the role of glutamate in mediating ipRGC light inputs into the SCN, we crossed mice in which Cre-recombinase expression is driven by the melanopsin promotor (Opn4(Cre/+)) with mice in which the second exon of VGLUT2 is flanked by loxP sites (VGLUT2(fl/fl)), producing ipRGCs that are unable to package glutamate into synaptic vesicles. Such mice had free-running circadian rhythms that did not entrain to a 12:12 light-dark (12:12 LD) cycle, nor did they show a phase delay after a 45-min light pulse administered at circadian time (CT) 14. A small subset of the mice did appear to entrain to the 12:12 LD cycle with a positive phase angle to lights-off; a similar entrainment pattern could be achieved in free-running mice if they were exposed to a 12:12 LD cycle with light of a greater intensity. Glutamate transmission from the ipRGCs is necessary for normal light entrainment of the SCN at moderate (0.35 W/m(2)) light levels, but residual transmission (possibly by PACAP in ipRGCs or by other RGCs) can weakly entrain animals, particularly at very high (6.53 W/m(2)) light levels, although it may be less effective at suppressing locomotor activity (light masking).

Background

Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZ^GABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration.

New methods

The present study is designed to validate the use of alternative DREADDs ligands-deschloroclozapine (DCZ) and compound 21 (C21)-and a new administration route, the voluntary oral administration.

Results

We show that IP injections of DCZ and C21 dose-dependently enhance SWS in PZ^GABA-hM3Dq mice, similar to CNO. We also show that oral administration of CNO, DCZ and C21 induces the same sleep phenotype as compared with IP injection.

Comparison with existing methods and conclusion

Therefore, DCZ and C21 are powerful alternatives to the use of CNO. Moreover, the voluntary oral administration is suitable for repeated dosing of DREADDs ligands.

Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

The suprachiasmatic nucleus is the circadian pacemaker of the mammalian brain. Suprachiasmatic nucleus neurons display synchronization of their firing frequency on a circadian timescale, which is required for the pacemaker function of the suprachiasmatic nucleus. However, the mechanisms by which suprachiasmatic nucleus neurons remain synchronized in vivo are poorly understood, although synaptic communication is considered indispensable. Suprachiasmatic nucleus neurons contain the neurotransmitter GABA and express GABA receptors. This has inspired the hypothesis that GABA signalling may play a central role in network synchronization, although this remains untested in vivo. Here, using local genetic deletion, we show that disruption of GABA synaptic transmission within the suprachiasmatic nucleus of adult mice results in the eventual deterioration of physiological and behavioural rhythmicity in vivo and concomitant cellular desynchrony in vitro. These findings suggest that intercellular GABA signalling is essential for behavioural rhythmicity and cellular synchrony of the suprachiasmatic nucleus neural network.